Suramin is one of the oldest drugs in use today. It is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense, and it is also used for surra in camels caused by Trypanosoma evansi. Yet despite one hundred years of use, suramin's mode of action is not fully understood. Suramin is a polypharmacological molecule that inhibits diverse proteins. Here we demonstrate that a DNA helicase of the pontin/ruvB-like 1 family, termed T. brucei RuvBL1, is involved in suramin resistance in African trypanosomes. Bloodstream-form T. b. rhodesiense under long-term selection for suramin resistance acquired a homozygous point mutation, isoleucine-312 to valine, close to the ATP binding site of T. brucei RuvBL1. The introduction of this missense mutation, by reverse genetics, into drug-sensitive trypanosomes significantly decreased their sensitivity to suramin. Intriguingly, the corresponding residue of T. evansi RuvBL1 was found mutated in a suramin-resistant field isolate, in that case to a leucine. RuvBL1 (Tb927.4.1270) is predicted to build a heterohexameric complex with RuvBL2 (Tb927.4.2000). RNAi-mediated silencing of gene expression of either T. brucei RuvBL1 or RuvBL2 caused cell death within 72 h. At 36 h after induction of RNAi, bloodstream-form trypanosomes exhibited a cytokinesis defect resulting in the accumulation of cells with two nuclei and two or more kinetoplasts. Taken together, these data indicate that RuvBL1 DNA helicase is involved in suramin action in African trypanosomes.

• Keywords
• Drug target, Drug-resistance, RuvB-like 1 DNA helicase, Suramin, Trypanosoma brucei, Trypanosoma evansi,
• MeSH
• DNA Helicases genetics   MeSH
• Suramin pharmacology   therapeutic use   MeSH
• Trypanosoma brucei brucei * genetics   MeSH
• Trypanosoma brucei rhodesiense genetics   MeSH
• Trypanosoma * genetics   MeSH
• Trypanosomiasis, African * drug therapy   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Helicases MeSH
• Suramin MeSH

Camels are considered an important food source in North Africa. Trypanosomiasis in camels is a life-threatening disease that causes severe economic losses in milk and meat production. Therefore, the objective of this study was to determine the trypanosome genotypes in the North African region. Trypanosome infection rates were determined by microscopic examination of blood smears and polymerase chain reaction (PCR). In addition, total antioxidant capacity (TAC), lipid peroxides (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were determined in erythrocyte lysate. Furthermore, 18S amplicon sequencing was used to barcode and characterizes the genetic diversity of trypanosome genotypes in camel blood. In addition to Trypanosoma, Babesia and Thelieria were also detected in the blood samples. PCR showed that the trypanosome infection rate was higher in Algerian samples (25.7%) than in Egyptian samples (7.2%). Parameters such as MDA, GSH, SOD and CAT had significantly increased in camels infected with trypanosomes compared to uninfected control animals, while TAC level was not significantly changed. The results of relative amplicon abundance showed that the range of trypanosome infection was higher in Egypt than in Algeria. Moreover, phylogenetic analysis showed that the Trypanosoma sequences of Egyptian and Algerian camels are related to Trypanosoma evansi. Unexpectedly, diversity within T. evansi was higher in Egyptian camels than in Algerian camels. We present here the first molecular report providing a picture of trypanosomiasis in camels, covering wide geographical areas in Egypt and Algeria.

• MeSH
• Antioxidants MeSH
• Phylogeny MeSH
• Genotype MeSH
• Superoxide Dismutase genetics   MeSH
• Trypanosoma * genetics   MeSH
• Trypanosomiasis * epidemiology   veterinary   MeSH
• Camelus MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Africa, Northern MeSH
• Names of Substances
• Antioxidants MeSH
• Superoxide Dismutase MeSH

Mitochondrial F-type adenosine triphosphate (ATP) synthases are commonly introduced as highly conserved membrane-embedded rotary machines generating the majority of cellular ATP. This simplified view neglects recently revealed striking compositional diversity of the enzyme and the fact that in specific life stages of some parasites, the physiological role of the enzyme is to maintain the mitochondrial membrane potential at the expense of ATP rather than to produce ATP. In addition, mitochondrial ATP synthases contribute indirectly to the organelle's other functions because they belong to major determinants of submitochondrial morphology. Here, we review current knowledge about the trypanosomal ATP synthase composition and architecture in the context of recent advances in the structural characterization of counterpart enzymes from several eukaryotic supergroups. We also discuss the physiological function of mitochondrial ATP synthases in three trypanosomatid parasites, Trypanosoma cruzi, Trypanosoma brucei and Leishmania, with a focus on their disease-causing life cycle stages. We highlight the reversed proton-pumping role of the ATP synthase in the T. brucei bloodstream form, the enzyme's potential link to the regulation of parasite's glycolysis and its role in generating mitochondrial membrane potential in the absence of mitochondrial DNA.

• Keywords
• ATP synthase, cryo-EM, mitochondria, mitochondrial membrane potential, oxidative phosphorylation,
• MeSH
• Genetic Engineering * MeSH
• Leishmania enzymology   MeSH
• Membrane Potential, Mitochondrial MeSH
• Mitochondrial Proton-Translocating ATPases genetics   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Trypanosoma brucei brucei enzymology   MeSH
• Trypanosoma cruzi enzymology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Mitochondrial Proton-Translocating ATPases MeSH
• Protozoan Proteins MeSH

The use of plant-derived natural products for the treatment of tropical parasitic diseases often has ethnopharmacological origins. As such, plants grown in temperate regions remain largely untested for novel anti-parasitic activities. We describe here a screen of the PhytoQuest Phytopure library, a novel source comprising over 600 purified compounds from temperate zone plants, against in vitro culture systems for Plasmodium falciparum, Leishmania mexicana, Trypanosoma evansi and T. brucei. Initial screen revealed 6, 65, 15 and 18 compounds, respectively, that decreased each parasite's growth by at least 50% at 1-2 µM concentration. These initial hits were validated in concentration-response assays against the parasite and the human HepG2 cell line, identifying hits with EC50 < 1 μM and a selectivity index of >10. Two sesquiterpene glycosides were identified against P. falciparum, four sterols against L. mexicana, and five compounds of various scaffolds against T. brucei and T. evansi. An L. mexicana resistant line was generated for the sterol 700022, which was found to have cross-resistance to the anti-leishmanial drug miltefosine as well as to the other leishmanicidal sterols. This study highlights the potential of a temperate plant secondary metabolites as a novel source of natural products against tropical parasitic diseases.

• Keywords
• African trypanosomiasis, Surra, drug discovery, leishmaniasis, malaria, natural products, neglected tropical diseases, temperate zone,
• Publication type
• Journal Article MeSH

Euglenozoa is a species-rich group of protists, which have extremely diverse lifestyles and a range of features that distinguish them from other eukaryotes. They are composed of free-living and parasitic kinetoplastids, mostly free-living diplonemids, heterotrophic and photosynthetic euglenids, as well as deep-sea symbiontids. Although they form a well-supported monophyletic group, these morphologically rather distinct groups are almost never treated together in a comparative manner, as attempted here. We present an updated taxonomy, complemented by photos of representative species, with notes on diversity, distribution and biology of euglenozoans. For kinetoplastids, we propose a significantly modified taxonomy that reflects the latest findings. Finally, we summarize what is known about viruses infecting euglenozoans, as well as their relationships with ecto- and endosymbiotic bacteria.

• Keywords
• Diplonemida, Euglenida, Kinetoplastida, microbial eukaryotes, phylogeny, systematics,
• MeSH
• Ecosystem MeSH
• Euglenozoa classification   genetics   physiology   virology   MeSH
• Phylogeny MeSH
• Mimiviridae pathogenicity   MeSH
• Symbiosis MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei, the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the FoF1-ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome FoF1-ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of FoF1-ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased FoF1-ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei FoF1-ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of FoF1-ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of FoF1-ATP synthase loss in insect versus mammalian forms of the parasite.

• Keywords
• ATP synthase, ATPase, Trypanosoma brucei, alternative oxidase, bioenergetics, electron transport, mitochondria, mitochondrial membrane potential, oxidative phosphorylation, respiration,
• MeSH
• Adenosine Triphosphate genetics   metabolism   MeSH
• Cell Cycle * MeSH
• Energy Metabolism * MeSH
• Membrane Potential, Mitochondrial MeSH
• Mitochondria genetics   metabolism   MeSH
• Proton-Translocating ATPases deficiency   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Proton-Translocating ATPases MeSH
• Protozoan Proteins MeSH

A recently redescribed two-flagellar trypanosomatid Vickermania ingenoplastis is insensitive to the classical inhibitors of respiration and thrives under anaerobic conditions. Using genomic and transcriptomic data, we analyzed its genes of the core metabolism and documented that subunits of the mitochondrial respiratory complexes III and IV are ablated, while those of complexes I, II, and V are all present, along with an alternative oxidase. This explains the previously reported conversion of glucose to acetate and succinate by aerobic fermentation. Glycolytic pyruvate is metabolized to acetate and ethanol by pyruvate dismutation, whereby a unique type of alcohol dehydrogenase (shared only with Phytomonas spp.) processes an excess of reducing equivalents formed under anaerobic conditions, leading to the formation of ethanol. Succinate (formed to maintain the glycosomal redox balance) is converted to propionate by a cyclic process involving three enzymes of the mitochondrial methyl-malonyl-CoA pathway, via a cyclic process, which results in the formation of additional ATP. The unusual structure of the V. ingenoplastis genome and its similarity with that of Phytomonas spp. imply their relatedness or convergent evolution. Nevertheless, a critical difference between these two trypanosomatids is that the former has significantly increased its genome size by gene duplications, while the latter streamlined its genome.

• Keywords
• Phytomonas, Vickermania ingenoplastis, genome sequencing, metabolism,
• Publication type
• Journal Article MeSH

Receptor adenylate cyclases (RACs) on the surface of trypanosomatids are important players in the host-parasite interface. They detect still unidentified environmental signals that affect the parasites' responses to host immune challenge, coordination of social motility, and regulation of cell division. A lesser known class of oxygen-sensing adenylate cyclases (OACs) related to RACs has been lost in trypanosomes and expanded mostly in Leishmania species and related insect-dwelling trypanosomatids. In this work, we have undertaken a large-scale phylogenetic analysis of both classes of adenylate cyclases (ACs) in trypanosomatids and the free-living Bodo saltans. We observe that the expanded RAC repertoire in trypanosomatids with a two-host life cycle is not only associated with an extracellular lifestyle within the vertebrate host, but also with a complex path through the insect vector involving several life cycle stages. In Trypanosoma brucei, RACs are split into two major clades, which significantly differ in their expression profiles in the mammalian host and the insect vector. RACs of the closely related Trypanosoma congolense are intermingled within these two clades, supporting early RAC diversification. Subspecies of T. brucei that have lost the capacity to infect insects exhibit high numbers of pseudogenized RACs, suggesting many of these proteins have become redundant upon the acquisition of a single-host life cycle. OACs appear to be an innovation occurring after the expansion of RACs in trypanosomatids. Endosymbiont-harboring trypanosomatids exhibit a diversification of OACs, whereas these proteins are pseudogenized in Leishmania subgenus Viannia. This analysis sheds light on how ACs have evolved to allow diverse trypanosomatids to occupy multifarious niches and assume various lifestyles.

• Keywords
• Kinetoplastida, adenylate cyclase, oxygen, phylogenetics, receptor, trypanosomatid,
• MeSH
• Adenylyl Cyclases genetics   MeSH
• Gene Duplication MeSH
• Phylogeny * MeSH
• Genome, Protozoan MeSH
• Host-Pathogen Interactions genetics   MeSH
• Evolution, Molecular * MeSH
• Trypanosomatina enzymology   genetics   MeSH
• Up-Regulation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylyl Cyclases MeSH

BACKGROUND: The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. RESULTS: We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. CONCLUSIONS: The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.

• Keywords
• Comparative genomics, Diplonemea, Euglenida, Evolution, Kinetochores, Kinetoplastea, Metabolism, Trypanothione,
• MeSH
• Biological Evolution * MeSH
• Euglenida genetics   metabolism   MeSH
• Euglenozoa genetics   metabolism   MeSH
• Genome, Protozoan * MeSH
• Kinetoplastida genetics   metabolism   MeSH
• Evolution, Molecular MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.

• Keywords
• DNA content, kinetoplast DNA, mitochondrial DNA, protist,
• MeSH
• Euglenozoa genetics   MeSH
• Phylogeny MeSH
• Kinetoplastida genetics   MeSH
• DNA, Mitochondrial genetics   isolation & purification   ultrastructure   MeSH
• Mitochondria genetics   MeSH
• Trans-Splicing genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Mitochondrial MeSH